U.S. patent number 5,110,725 [Application Number 07/680,978] was granted by the patent office on 1992-05-05 for optical probe for the cytochrome p-450 cholesterol side chain cleavage enzyme.
This patent grant is currently assigned to The United States of America as represented by the United States. Invention is credited to Babetta L. Marrone, Daniel J. Simpson, Clifford J. Unkefer, Thomas W. Whaley.
United States Patent |
5,110,725 |
Marrone , et al. |
May 5, 1992 |
Optical probe for the cytochrome P-450 cholesterol side chain
cleavage enzyme
Abstract
An optical probe enables the study of enzyme activity by
absorbance spectroscopy or by sensitive fluorescence methods. In
particular, the probe provides the ability to monitor the activity
of cytochrome P-450.sub.scc enzyme, the rate limiting enzyme for
steroid biosynthesis. Located on the inner mitochondrial membrane,
P-450.sub.scc catalyzes the conversion of cholesterol to
pregnenolone and isocapraldehyde by sequential oxidations of the
cholesterol side chain. The fluorogenic probe includes a
cholesterol-like steroid linked to a chromophore through a linking
group. The chromophore is selected to have little optical response
when linked to the steroid substrate and an enhanced optical
response when cleaved from the substrate and linking group. Thus, a
fluorescent anion that can be optically detected is generated by
the side-chain cleavage reaction during steroidogenesis.
Inventors: |
Marrone; Babetta L. (Los
Alamos, NM), Simpson; Daniel J. (Los Alamos, NM),
Unkefer; Clifford J. (Los Alamos, NM), Whaley; Thomas W.
(Santa Fe, NM) |
Assignee: |
The United States of America as
represented by the United States (Washington, DC)
|
Family
ID: |
24733274 |
Appl.
No.: |
07/680,978 |
Filed: |
April 5, 1991 |
Current U.S.
Class: |
435/11;
435/189 |
Current CPC
Class: |
C12Q
1/60 (20130101) |
Current International
Class: |
C12Q
1/60 (20060101); C12Q 001/60 (); C12N 009/02 () |
Field of
Search: |
;435/11,189 |
Other References
R B. Hochberg et al., "A Simple and Precise Assay of the Enzymatic
Conversion of Cholesterol into Pregnenolone", 13 Biochem. No. 3,
pp. 603-608 (1974). .
N. B. Goldring et al., "Immunofluorescent Probing of the
Mitochondrial Cholesterol Side-Chain Cleavage Cytochrome P-450
Expressed in Differentiating Granulossa Cells in Culture", 119
Endocrinology, No. 6, pp. 2821-2832 (1986). .
J. D. Lambeth, "Cytochrome P-450.sub.scc --A Review of the
Specificity and Properties of the Cholesterol Binding Site", 12
Endocrine Research, No. 4, pp. 371-392 (1986). .
Kao et al., Biochemistry 17:2689-2696 (78). .
Drew, J. et al., J. Org. Chem. 52:4047-4052 (87), Synthesis from
Pregnenolone of Fluorescent Cholesterol Analogue Probes with
Conjugated Unsaturation in the Side Chain. .
Babb, B. et al., CA 109(1):3045Y (87), Hydrolyzable Fluorescent
Substrates and Analytical Determinations of Enzymes Using
Same..
|
Primary Examiner: Naff; David M.
Assistant Examiner: Saucier; S.
Attorney, Agent or Firm: Wilson; Ray G. Gaetjens; Paul D.
Moser; William R.
Claims
What is claimed is:
1. A method for quantifying the activity of the P-450.sub.scc
enzyme in the conversion of cholesterol to pregnenolone in
steroidogenesis, comprising the steps of:
forming a fluorogenic probe having a cholesterol-based steroid
connected through a linking group at the C-22 position with a
chromophore effective to have a low optical response when attached
to said steroid and a high optical response as an anion, said
chromophore having a molecular size effective to not interfere with
an enzymatic reaction that cleaves said chromophore from said
steroid;
incorporating said probe in a process for said conversion of
cholesterol to pregnenolone;
reacting said probe with the P-450.sub.scc enzyme to cleave said
side-chain from the probe and form said anion having said high
optical response from said chromophore; and
exciting said anion to obtain said high optical response; and
optically detecting said response as a measure of said
P-450.sub.scc enzyme activity.
2. A method according to claim 1, wherein said optical response is
fluorescence.
3. A method according to claim 1, wherein said linking group is
selected from the group consisting of ##STR1##
4. A method according to claim 1, wherein said chromophore is
selected from the group consisting of ##STR2##
5. A method according to claim 3, wherein said chromophore is
selected from the group consisting of ##STR3##
6. A method according to claim 4, wherein said chromophore is
resorufin ether.
Description
BACKGROUND OF INVENTION
This invention relates to the study of enzymatic events in cells
and, more particularly, to the study of enzymatic events in single
cells by optical detection methods. This invention is the result of
a contract with the Department of Energy (Contract No.
W-7405-ENG-36).
Enzymatic events in cells include steroid biosynthesis.
Identification of such events provides important information for
ongoing research on factors regulating the growth and
differentiated function of steroidogenic cells. In one particular
event, the cytochrome P-450 cholesterol side-chain cleavage enzyme
(P-450.sub.scc) controls the rate-limiting step of steroidogenesis,
the enzymatic conversion of cholesterol to pregnenolone.
In one attempt to characterize the side-chain cleavage event, the
amount of P-450.sub.scc has been investigated by immunofluorescent
staining of P-450.sub.scc with an antibody to the P-450.sub.scc
enzyme. See N. B. Goldring et al., "Immunofluorescent Probing of
the Mitochondrial Cholesterol Side-Chain Cleavage Cytochrome P-450
Expressed in Differentiating Granulosa Cells in Culture," 119
Endocrinology, pp. 2821-2832 (1986). This technique, however, can
not measure P-450.sub.scc enzyme activity since the
immunofluorescent staining requires cell fixation.
One measure of the cleavage event is the amount of the side chain
fragment produced by the enzymatic cleavage. The enzymatic
conversion of cholesterol to pregnenolone by the P-450.sub.scc
enzyme can be measured from the amount of radioisotopically labeled
isocapraldehyde formed from cholesterol when the sterol substrate
bears a radioisotope on the side chain. See R. B. Hochberg et al.,
"A Simple and Precise Assay of the Enzymatic Conversion of
Cholestrol in Pregnenolone," 13 Biochemistry, No. 13, pp. 603-608
(1974).
It would be desirable, however, to use a rapid, sensitive, single
cell analysis technique, such as fluorescence detection in flow
cytometry, for the study of these metabolic events. However,
fluorescent probes have not been available that are specific to a
particular cell function, such as the activity of a key enzyme.
This problem is addressed by the present invention and a
fluorescent probe is developed that is specific for identification
of the rate-limiting cholesterol side-chain cleavage by the
P-450.sub.scc enzyme.
Accordingly, it is an object of the present invention to provide a
fluorescent probe to measure the activity of the enzyme responsible
for the conversion of cholesterol to other steroids.
It is another object of the present invention to provide a steroid
probe that is non-fluorescent until cleaved by P-450.sub.scc.
Additional objects, advantages and novel features of the invention
will be set forth in part in the description which follows, and in
part will become apparent to those skilled in the art upon
examination of the following or may be learned by practice of the
invention. The objects and advantages of the invention may be
realized and attained by means of the instrumentalities and
combinations particularly pointed out in the appended claims.
SUMMARY OF THE INVENTION
To achieve the foregoing and other objects, and in accordance with
the purposes of the present invention, as embodied and broadly
described herein, the apparatus of this invention may comprise a
probe for use in quantifying the activity of the P-450.sub.scc
enzyme in steroidogenesis. The probe is a fluorogenic substrate for
the P-450.sub.scc enzyme having a cholesterol-like steroid with the
side chain at the C-22 position replaced with a chromophore
selected from the xanthene dye group.
In another characterization of the present invention, a method for
quantifying the activity of the P-450.sub.scc enzyme allows the
rate-limiting process in steroidogenesis, the conversion of
cholesterol to pregnenolone, to be examined. A fluorogenic probe is
a cholesterol-like steroid with the side-chain at the C-22 position
replaced with the chromophore resorufin ether. The side chain is
cleaved from the probe by the P-450.sub.scc enzyme to generate the
steroid pregnenolone and a highly fluorescent resorufin anion. The
presence of the resorufin anion is thereafter detected by sensitive
optical detection methods, such as absorbance spectroscopy or
fluorescence detection used in flow and image cytometry, to measure
P-450.sub.scc expression and regulation in cells or subcellular
fractions or with a purified or semi-purified enzyme
preparation.
In another characterization of the present invention, a process is
provided for synthesizing a fluorescent substrate for use in
quantifying activity of the P-450.sub.scc enzyme. The process
includes the following steps:
a. treating 3.beta.-acetoxy-22,23-bisnor-5-cholenic acid with
thionyl chloride to yield 3.beta.-acetoxy-22,23-bisnor-5-cholenyl
chloride;
b. reducing said acid chloride with lithium
tri-tertbutoxyaluminohydride to yield the alcohol
3.beta.-acetoxy-5-cholene-22-ol;
c. treating said alcohol with p-toluenesulfonyl chloride in
pyridine to yield the tosylate
3.beta.-acetoxy-22-p-toluenesulfonyl-5-cholenate;
d. dissolving said tosylate in dimethyl sulfoxide and treating with
an excess of resorufin sodium salt at temperatures between
55.degree.-70.degree. C. to yield the product
3.beta.-acetoxy-22-phenoxazonoxy-5-cholene;
e. separating said product by chromatography and hydrolysis with
KOH/methanol to yield said fluorogenic substrate
22-phenoxazonoxy-5-cholene-3.beta.-ol; and
f. separating said fluorogenic substrate by chromatography on
silica.
BRIEF DESCRIPTION OF THE DRAWINGS
The accompanying drawings, which are incorporated in and form a
part of the specification, illustrate an embodiment of the present
invention and, together with the description, serve to explain the
principles of the invention. In the drawings:
FIGS. 1A-C illustrate the chemical composition of optical probes
according to the present invention.
FIG. 2 illustrates the side-chain cleavage of a cholesterol-like
steroid by the P-450.sub.scc enzyme.
FIG. 3 illustrates the chemical reactions to generate a fluorescent
probe according to one embodiment of the present invention.
DETAILED DESCRIPTION OF THE INVENTION
In accordance with the present invention, an optical probe enables
the study of enzyme activity in single steroidogenic cells by
absorbance spectroscopy or by sensitive fluorescence methods. In
particular, the probe provides the ability to monitor in real time
the activity of cytochrome P-450.sub.scc enzyme, the rate limiting
enzyme for steroid biosynthesis. Located on the inner mitochondrial
membrane, P-450.sub.scc catalyzes the conversion of cholesterol to
pregnenolone and isocapraldehyde by sequential oxidations of the
cholesterol side chain.
FIGS. 1A-C depict the chemical composition of the components of an
optical probe according to the present invention. A
cholesterol-like steroid is linked to a chromophore through a
linking group. In order to bind to the enzyme, the steroid
substrate, FIGS. 1A and 1B, has a 3.beta.-hydroxy group,
unsaturation in the 5-6 bond, and has cholesterol-like
stereochemistry at the C-17 and C-20.alpha. positions. The R
position can be filled from the group consisting of H, alkyl, and
alkyl--CO--. As used herein, the term alkyl is limited to the low
alkyl groups with 1-6 carbon atoms, i.e., methyl, ethyl, isopropyl,
etc.
The chromophore is selected to have advantageous spectral
properties, i.e., to have little optical response when linked to
the steroid substrate and an enhanced optical response when cleaved
from the substrate and linking group. The chromophore molecule
should also be of modest size to not interfere with the side chain
cleavage reaction. A preferred chromophore is selected from the
xanthene dyes, linked as shown in FIG. 1A or 1B, where R.sub.2 is
H, halogen, alkyl, --O--alkyl, --COOalkyl, or --COOH; X is O, S,
NH, or N-alkyl; and Z is O or N,N-dialkyl.
The structural requirements of the linking group, FIG. 1C, are not
stringent, where the X' position can be filled from either O or S.
Any one of the three linking groups may be selected, with a
preferred link being the simple O or S link. The group containing
an intermediate C provides a somewhat longer link and the group
containing the (CH.sub.2).sub.n provides longer linker arms. All
three linking groups are expected to be cleaved with the
P-450.sub.scc enzyme and to not interfere with the generation of
the active chromophore.
Referring now to FIG. 2, the P-450.sub.scc probe 10 is a
cholene-based steroid covalently conjugated at the C-22 position to
resorufin. As a result of this conjugation the resorufin
fluorescence is shifted and has a 40-fold lower quantum efficiency
than the resorufin anion. Specificity of probe 10 for the
P-450.sub.scc enzyme is achieved by incorporating known structural
and stereochemical features to the cholene-ring for enzyme
recognition. Generation of a fluorescent signal is obtained by
release of the resorufin moiety 16 by the side chain cleavage
enzyme.
The mechanics of P-450sc enzyme-substrate (probe 10) binding and
side chain cleavage 12 are well known. Once a substrate 10, such as
cholesterol, is recognized and positioned into the enzyme active
site, side chain cleavage by P-450.sub.scc 12 proceeds in three
oxidative steps: 1) hydroxylation of C-22 at the pro-R methylene
position; 2) hydroxylation of the adjacent 20.alpha.-methine
position to give a vicinal diol intermediate; and 3) oxidative
cleavage of the diol to give pregnenolone 14 and a side chain
fragment 16. In order to bind to the enzyme, a steroid substrate
should contain a 3.beta.-hydroxy group, unsaturation in the 5-6
bond, and have cholesterol-like stereochemistry at the C-17 and
C-20.alpha. positions. The structural requirements of the side
chain beyond the C-22 position are less stringent.
Resorufin (7-hydroxy-3H-phenoxazin-3-one) was chosen as a
fluorogenic reporter because of its advantageous spectral
properties and its modest size. Covalently conjugated through its
oxy anion, resorufin shows a large increase in absorbance energy
(approximately 110 nm), a drop in absorbance extinction (by
approximately 1/3) relative to the anion, and a 40-fold difference
in fluorescence quantum yield. Presumably, metabolism of a
resorufin ether 10 by P-450.sub.scc proceeds by hydroxylation at
the C-1 position of the ether side chain, to yield a resorufin
hemiacetal that undergoes cleavage to afford the highly fluorescent
resorufin anion. The fluorescence signal generated by the formation
of resorufin anion is a quantitative indicator of enzyme
activity.
Synthesis of fluorogenic substrate probe 10,
22-phenoxazonoxy-5-cholene-3.beta.-ol, is shown in the scheme
depicted in FIG. 3. In step I, 3.beta.-acetoxy-22,
23-bisnor-5-cholenic acid 20 (1.0 g, Steraloids, Inc., Wilton,
N.H.) was dissolved in methylene chloride (5 mL) and cooled to
0.degree.-5.degree. C. with an ice bath. The selected R group was
alkyl--CO--, and particularly tert-butoxy. Thionyl chloride (900
mg) was added dropwise via a syringe to the clear solution. The
solution was stoppered, allowed to warm to room temperature, and
stirred for 20 h. This mixture was diluted with methylene chloride,
extracted with aqueous NaCl and dried and evaporated to a white
solid.
This crude acid chloride was dissolved in methylene chloride (5 mL)
and cooled to -65.degree. C. (dry ice/acetone). Lithium
tri-tert-butoxyaluminhydride in tetrahydrofuran (1.0M, 2.3 mL) was
added under argon to the acid chloride over 20 min. The reaction
was kept at -65.degree. C. for 1 h after addition, then allowed to
warm to room temperature. Excess hydride reagent was destroyed by
the slow dropwise addition of water. The mixture was extracted with
methylene chloride. The organic layer was washed extensively with
1.0 N HCl, aqueous NaCl, and dried and evaporated. Compound 22,
3.beta.-acetoxy-5-cholene-22-ol, was purified by column
chromatography eluting with 1% methanol/methylene chloride and
obtained as a solid (640 mg) from methylene chloride/n-hexane.
In step II, compound 22 was dissolved in dry pyridine (1 mL) in a
small reacta-vial and p-toluenesulfonyl chloride (175 mg) was
added. The vial was capped and stirred for 20 h. This mixture was
diluted with ether, washed with 0.1 N HCl (3.times.50 mL), and
dried and evaporated to a colorless oil. Compound 24 (185 mg),
3.beta.-acetoxy-22-p-toluenesulfonyl-5-cholene, was recovered as a
crystalline white solid from ether/n-hexane. For step III, steroid
24(240 mg) was suspended in dimethyl sulfoxide (3 mL) and combined
with resorufin (200 mg). This mixture was stirred under argon at
55.degree. C. for 10 days. The dimethyl sulfoxide was removed by
lyophylization (10 h, 10.sup.-5 torr). The resulting solid was
suspended in methylene chloride and filtered to remove unreacted
resorufin and a bright orange material. Thin layer chromatography
showed one orange spot, more polar than the tosylate precursor, as
seen after development with phosphomolybdic acid. Compound 24 was
purified by flash chromatography (eluting with 3%
tetrahydrofuran/methylene chloride) and obtained as small orange
needles from methylene chloride/n-hexane. This process yielded 22
mg of compound 26, with 140 mg of compound 24 remaining unreacted.
The unreacted compound 24 can be recycled through step III to
produce additional compound 26.
The desired fluorogenic probe 10 was then generated in step IV.
Acetate compound 26 was suspended in 5% KOH/methanol (2 mL),
containing approximately 1% water, and refluxed for 30 min while
argon was bubbled through the solution. After cooling, methylene
chloride was added. This mixture was washed with 1 M HCl, water,
and was dried and evaporated. Thin layer chromatography (TLC)
showed two spots, one minor spot (R.sub.f =0.5) of compound 26 and
the more polar product (R.sub.f =0.2) 10. Because of the small
amount of material (yield of 7 mg), purification was achieved by
preparative-scale TLC eluting with 3% methanol/methylene chloride
and the product recovered was obtained as an orange solid from
methylene chloride/n-hexane. It will be appreciated that the above
protocol may generally be used to synthesize fluorogenic probes
from starting materials appropriate to the chemical groups depicted
in FIG. 1.
Fluorogenic probe 10 has been shown to be selective for
P-450.sub.scc activity in intact mitochondria and cells. A
saturated stock solution of probe 10 was made in 95% ethanol and
filtered with a 0.2 .mu.m filter to a concentration of 100 .mu.M.
Probe 10 was added at 1:50 or 1:100 dilutions to cell or
mitochondria suspensions.
Cell Preparations: Granulosa cell layers were collected separately
from the 5 largest preovulatory follicles of laying hens and cells
were dispersed by collagenase digestion (Endocrinology
122:651-658). The cell concentration was adjusted to
1.times.10.sup.6 cells/mL in modified medium 199 (no phenol red;
added 25 mM HEPES, 0.35 g/L sodium bicarbonate, 100 mg/L
1-glutamate, and 1 g/L bovine serum albumin). The MA-10 cells
(Endocrinology 108:88-95) were grown in RPMI-1640 medium
supplemented with 15% horse serum and used three days after
plating. Cell cultures of the Chinese hamster ovary (CHO) cell line
were grown in spinner flasks with Ham's F-10 medium supplemented
with 15% bovine calf serum. Media were obtained from Gibco BRL
(Gaithersburg, Md.) and sera were obtained from HyClone
Laboratories (Logan, Utah). Except where indicated all other
reagents for cell preparations were obtained from Sigma Chemical
Co. (St. Louis, Mo.). Cells in suspension (1.times.10.sup.6 /mL)
were kept on ice prior to use and at 37.degree. C. during all
incubations with probe 10.
Spectrofluorometry: Fluorescence emission measurements were
obtained on a SPEX Fluorolog-II spectrofluorometer (SPEX
Industries, Inc., Edison, N.J.) fitted with a thermostatted cuvette
holder and interfaced to an IBM-AT computer for data collection and
processing. Resorufin fluorescence was measured from 550-700 nm (8
nm slit width) with 530 nm excitation (10 nm slit width). Spectra
were background-corrected by spectral subtraction of the initial
(zero) time point. Samples were incubated at 37.degree. C. during
the experiment.
Flow Cytometry: For microspectrofluorometric demonstration the
Fourier transform flow cytometer (FTCS-1) at Los Alamos National
Laboratory (SPIE Proceedings, Bioimaging and Two-Dimensional
Spectroscopy, 1205:126-133) was used to analyze spectral changes in
the P-450.sub.scc probe fluorescence over time. A single argon
laser tuned to 514 nm was used to excite both the substrate and
product. The spectrum of the expected fluorescent product,
resorufin, with an emission maximum of 588 nm, and the spectrum of
the fluorogenic substrate, with an emission maximum of 562 nm were
obtained from each sample.
The specificity of the probe for the P-450.sub.scc enzyme was
demonstrated initially on a spectrofluorometer using mitochondrial
preparations from chicken granulosa and MA-10 cells. Both time (30
min to 5 h) and substrate concentration-dependent (from 0.01 to 2
.mu.M) resorufin production was observed with these steroidogenic
mitochondria. The addition of known inhibitors of P-450.sub.scc
enzymatic activity (aminoglutethimide and ketoconazole) attenuated
the observed fluorescence. Mitochondrial preparations from
nonsteroidogenic CHO cells showed no production of
fluorescence.
To confirm cellular metabolism, both MA-10 and granulosa cell
suspensions were incubated with varying concentrations of the probe
substrate. Fluorescence emission was monitored at discrete times
from 0-4 h. A linear increase in resorufin fluorescence was
observed over time and was associated with the cellular metabolism
of the probe by the P-450.sub.scc enzyme. Based on the amount of
fluorescence in a typical cell incubation the cumulative conversion
of the probe was in the concentration range of 100-300 pM. After
long (3-5 h) incubations of granulosa or MA-10 cells with a high
concentration (1-2 .mu.M) of the P-450.sub.scc probe, a two-fold
accumulation of progesterone over the background progesterone
content of the cells was measured by specific progesterone
radioimmunoassay (Endocrine Sciences, Tarzana, Calif.).
The Fourier transform flow cytometer was used to track the
metabolism of the fluorogenic substrate in individual granulosa
cells. The spectral information obtained on a cell-by-cell basis is
used to resolve fluorochromes in the cells with highly overlapping
emission spectra. Using this instrument, cellular fluorescence of
the substrate is resolved from the cellular fluorescence of the
product, resorufin anion, based on their unique spectral
characteristics during the uptake and metabolism of the
P-450.sub.scc probe.
By monitoring spectral changes in the cellular fluorescence at
discrete time points from 30 min to 5 h with the FTCS-1 an
increased metabolism of the substrate to the product resorufin was
observed. The slope of the cell distribution shifted from one that
was aligned primarily with the substrate to one that was aligned
essentially 100% with the product resorufin after 5 h. A linear
increase in the average ratio of the two fluorescent components
(substrate and resorufin) was observed in the accumulation of
resorufin fluorescence over time. By comparison, in experiments
using nonsteroidogenic CHO cells, there was no evidence of
significant metabolism of the substrate to resorufin. Following a 3
h incubation of the P-450.sub.scc substrate with CHO cells,
cellular fluorescence remained primarily consistent with the
substrate spectrum.
Conventional flow cytometric analysis of P-450.sub.scc activity was
demonstrated in a two-laser experiment on a multiparameter flow
cytometer in which green (substrate) fluorescence and red
(resorufin) fluorescence were measured at discrete time intervals
from 5 min to 2 h after allowing granulosa cells to react with the
P-450.sub.scc probe at 37.degree. C. Uptake of the substrate (green
fluorescence) plateaued at 30 minutes, presumably as the cells
equilibrated with the P-450.sub.scc probe in solution. By
comparison, product (red fluorescence) continued to increase in
cells during the incubation.
Thus, a mechanism-based, fluorogenic probe substrate for the
P-450.sub.scc enzyme is shown to be a sensitive, quantitative
indicator of P-450.sub.scc activity in populations of steroidogenic
cells using fluorescence detection methods. Moreover, preliminary
studies in granulosa cells have also shown that acute gonadotropin
treatment increases resorufin fluorescence due to metabolism of the
substrate, suggesting that the P-450.sub.scc probe can be used for
the study of enzyme regulation. The P-450.sub.scc probe, because of
its specificity and the sensitivity afforded by fluorescence
detection, should have widespread applicability to the study of
endocrine mechanisms regulating P-450.sub.scc enzyme activity
during various stages of growth, differentiation, and disease in
steroidogenic cell populations.
The foregoing description of an embodiment of the invention have
been presented for purposes of illustration and description. It is
not intended to be exhaustive or to limit the invention to the
precise form disclosed, and obviously many modifications and
variations are possible in light of the above teaching. The
embodiment was chosen and described in order to best explain the
principles of the invention and its practical application to
thereby enable others skilled in the art to best utilize the
invention in various embodiments and with various modifications as
are suited to the particular use contemplated. It is intended that
the scope of the invention be defined by the claims appended
hereto.
* * * * *